Background: We evaluated the effect of four blood storage conditions (refrigeration, fixation [with the Cyto-Chex and TransFix tubes], and physiologic stabilization using Ficoll polymer PM70) for 72 hours in flow cytometric analysis of paroxysmal nocturnal hemoglobinuria (PNH).
Methods: Peripheral whole blood (WB) was collected in Cyto-Chex tubes (Streck, USA), TransFix tubes (Cytomark, UK) and ethylenediaminetetraacetic acid (EDTA) tubes (Becton Dickinson, USA) from 20 patients with a PNH clone (≥1.0%) and 15 controls. PM70 (10%; GE Healthcare, USA)-EDTA WB was prepared. Samples were stored at room temperature (RT) or 4°C for 72 hours. Flow cytometric analysis was performed within 4 hours and 72 hours.
Results: The percentage of PNH clones was stable in stored WB at RT without systemic error, except for those stored in EDTA WB. A substantial correlation was observed between the fresh WB and stored WB. The mean fluorescence intensity of CD235a in red blood cells (RBCs) and CD15 in neutrophils decreased in stored WB, while CD59, CD24, and fluorescent aerolysin of the PNH clone showed no significant difference. The percentage of CD15 (+) neutrophils was not different between the fresh WB and stored WB. However, the viability of granulocytes was more than 90% in the refrigerated EDTA WB.
Conclusions: Stable measurement of PNH clones in RBCs and neutrophils is possible in refrigerated the EDTA WB for up to 72 hours. In the case of storage at RT, physiological stabilization using 10% PM70 can be an alternative method for WB fixation.

Keywords: Paroxysmal hemoglobinuria, Flow cytometry, Stabilization, Fluorescence